Glucopyranosyloxybenzylbenzene derivatives and medicinal compositions containing the same

ABSTRACT

The present invention relates to glucopyranosyloxy-benzylbenzene derivatives represented by the general formula:  
                 
wherein P represents a group forming a prodrug; and R represents a lower alkyl group, a lower alkoxy group, a lower alkylthio group, a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted (lower alkoxy) group or a lower alkoxy-substituted (lower alkylthio) group, which have an improved oral absorption and can exert an excellent inhibitory activity in human SGLT2 in vivo and which are useful as agents for the prevention or treatment of a disease associated with hyperglycemia such as diabetes, diabetic complications or obesity, and pharmaceutical compositions comprising the same.

TECHNICAL FIELD

The present invention relates to glucopyranosyloxy-benzylbenzenederivatives which are useful as medicaments and pharmaceuticalcompositions comprising the same.

More particularly, the present invention relates toglucopyranosyloxybenzylbenzene derivatives, which are useful as agentsfor the prevention or treatment of a disease associated withhyperglycemia such as diabetes, diabetic complications or obesity,represented by the general formula:

wherein P represents a group forming a prodrug; and R represents a loweralkyl group, a lower alkoxy group, a lower alkylthio group, a loweralkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted(lower alkoxy) group or a lower alkoxy-substituted (lower alkylthio)group, of which glucopyranosyl-oxybenzylbenzene derivatives, which havean inhibitory activity in human SGLT2, represented by the generalformula:

wherein R represents a lower alkyl group, a lower alkoxy group, a loweralkylthio group, a lower alkoxy-substituted (lower alkyl) group, a loweralkoxy-substituted (lower alkoxy) group or a lower alkoxy-substituted(lower alkylthio) group, are active forms, and relates to pharmaceuticalcompositions comprising the same.

BACKGROUND ART

Diabetes is one of lifestyle-related diseases with the background ofchange of eating habit and lack of exercise. Hence, diet and exercisetherapies are performed in patients with diabetes. Furthermore, when itssufficient control and continuous performance are difficult, drugtreatment is simultaneously performed. At present, biguanides,sulfonylureas and agents for reducing insulin resistance have beenemployed as antidiabetic agents. However, biguanides and sulfonylureasshow occasionally adverse effects such as lactic acidosis andhypoglysemia, respectively. In case of using agents for reducing insulinresistance, adverse effects such as edema occasionally are observed, andit is also concerned for advancing obesity. Therefore, in order to solvethese problems, it has been desired to develop antidiabetic agentshaving a new mechanism.

In recent years, development of new type antidiabetic agents has beenprogressing, which promote urinary glucose excretion and lower bloodglucose level by preventing excess glucose reabsorption at the kidney(J. Clin. Invest., Vol.79, pp.1510-1515 (1987)). In addition, it isreported that SGLT2 (Na⁺/glucose cotransporter 2) is present in the S1segment of the kidney's proximal tubule and participates mainly inreabsorption of glucose filtrated through glomerular (J. Clin. Invest.,Vol.93, pp.397-404 (1994)). Accordingly, inhibiting a human SGLT2activity prevents reabsorption of excess glucose at the kidney,subsequently promotes excreting excess glucose though the urine, andnormalizes blood glucose level. Therefore, fast development ofantidiabetic agents which have a potent inhibitory activity in humanSGLT2 and have a new mechanism has been desired. Furthermore, since suchagents promote the excretion of excess glucose though the urine andconsequently the glucose accumulation in the body is decreased, they arealso expected to have a preventing effect on obesity.

DISCLOSURE OF THE INVENTION

The present inventors have studied earnestly to find compounds having aninhibitory activity in human SGLT2. As a result, it was found thatcompounds represented by the above general formula (I) are converted invivo into their active forms, glucopyranosyloxybenzylbenzene derivativesrepresented by the above general formula (II), and show an excellentinhibitory activity in human SGLT2 as mentioned below, thereby formingthe basis of the present invention.

The present invention is to provide the followingglucopyranosyloxybenzylbenzene derivatives which exert an inhibitoryactivity in human SGLT2 in vivo and show an excellent hypoglycemiceffect by excreting excess glucose in the urine through preventing thereabsorption of glucose at the kidney, and pharmaceutical compositionscomprising the same.

This is, the present invention relates to aglucopyranosyloxybenzylbenzene derivative represented by the generalformula:

wherein P represents a group forming a prodrug; and R represents a loweralkyl group, a lower alkoxy group, a lower alkylthio group, a loweralkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted(lower alkoxy) group or a lower alkoxy-substituted (lower alkylthio)group.

The present invention relates to a pharmaceutical composition comprisingas an active ingredient a glucopyranosyloxybenzylbenzene derivativerepresented by the above general formula (I).

The present invention relates to a human SGLT2 inhibitor comprising asan active ingredient a glucopyranosyloxybenzylbenzene derivativerepresented by the above general formula (I).

The present invention relates to an agent for the prevention ortreatment of a disease associated with hyperglycemia, which comprises asan active ingredient a glucopyranosyloxybenzylbenzene derivativerepresented by the above general formula (I).

The present invention relates to a method for the prevention ortreatment of a disease associated with hyperglycemia, which comprisesadministering a therapeutically effective amount of aglucopyranosyloxybenzylbenzene derivative represented by the abovegeneral formula (I).

The present invention relates to a use of aglucopyranosyloxybenzylbenzene derivative represented by the abovegeneral formula (I) for the manufacture of a pharmaceutical compositionfor the prevention or treatment of a disease associated withhyperglycemia.

In the present invention, the term “prodrug” means a compound which isconverted into a glucopyranosyloxybenzylbenzene derivative representedby the above general formula (II) as an active form thereof in vivo. Asexamples of groups forming prodrugs, a hydroxy-protective group usedgenerally as a prodrug, such as a lower acyl group, a loweralkoxy-substituted (lower acyl) group, a loweralkoxycarbonyl-substituted (lower acyl) group, a lower alkoxycarbonylgroup and a lower alkoxy-substituted (lower alkoxycarbonyl) group, areillustrated.

Also, in the present invention, the term “lower alkyl group” means astraight-chained or branched alkyl group having 1 to 6 carbon atoms suchas a methyl group, an ethyl group, a propyl group, an isopropyl group, abutyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, apentyl group, an isopentyl group, a neopentyl group, a tert-pentylgroup, a hexyl group or the like; the term “lower alkoxy group” means astraight-chained or branched alkoxy group having 1 to 6 carbon atomssuch as a methoxy group, an ethoxy group, a propoxy group, an isopropoxygroup, a butoxy group, an isobutoxy group, a sec-butoxy group, atert-butoxy group, a pentyloxy group, an isopentyloxy group, aneopentyloxy group, a tert-pentyloxy group, a hexyloxy group or thelike; and the term “lower alkylthio group” means a straight-chained orbranched alkylthio group having 1 to 6 carbon atoms such as a methylthiogroup, an ethylthio group, a propylthio group, an isopropylthio group, abutylthio group, an isobutylthio group, a sec-butylthio group, atert-butylthio group, a pentylthio group, an isopentylthio group, aneopentylthio group, a tert-pentylthio group, a hexylthio group or thelike. The term “lower alkoxy-substituted (lower alkyl) group means theabove lower alkyl group substituted by the above lower alkoxy group; theterm “lower alkoxy-substituted (lower alkoxy) group means the abovelower alkoxy group substituted by the above lower alkoxy group; and theterm “lower alkoxy-substituted (lower alkylthio) group means the abovelower alkylthio group substituted by the above lower alkoxy group. Theterm “lower acyl group” means a straight-chained, branched or cyclicacyl group having 2 to 7 carbon atoms such as an acetyl group, apropionyl group, a butyryl group, an isobutyryl group, a pivaloyl group,a hexanoyl group and a cyclohexylcarbonyl group; and the term “loweralkoxy-substituted (lower acyl) group means the above lower acyl groupsubstituted by the above lower alkoxy group. The term “loweralkoxycarbonyl group” means a straight-chained, branched or cyclicalkoxycarbonyl group having 2 to 7 carbon atoms such as amethoxycarbonyl group, an ethoxycarbonyl group, an isopropyloxycarbonylgroup, an isobutyloxycarbonyl group and a cyclohexyloxycarbonyl group;the term “lower alkoxycarbonyl-substituted (lower acyl) group means theabove lower acyl group substituted by the above lower alkoxycarbonylgroup such as a 3-(ethoxycarbonyl)propionyl group; and the term “loweralkoxy-substituted (lower alkoxycarbonyl) group means the above loweralkoxycarbonyl group substituted by the above alkoxy group such as a2-methoxyethoxycarbonyl group.

In the substituent R, a lower alkyl group and a lower alkoxy group arepreferable, a straight-chained or branched alkyl group having 1 to 4carbon atoms and a straight-chained or branched alkoxy group having 1 to3 carbon atoms are more preferable, and an ethyl group and a methoxygroup are most preferable. In the substituent P, a lower acyl group anda lower alkoxycarbonyl group are preferable. As the lower acyl group, astraight-chained or branched acyl group having 4 to 6 carbon atoms ispreferable, and a butyryl group and a hexanoyl group are morepreferable. As the lower alkoxycarbonyl group, a straight-chained orbranched alkoxycarbonyl group having 2 to 5 carbon atoms is preferable,and a methoxycarbonyl group and an ethoxycarbonyl group are morepreferable.

The compounds of the present invention can be prepared by introducing ahydroxy-protective group being capable of using commonly in prodrugsinto the hydroxy group of a glucopyranosyloxybenzylbenzene derivativerepresented by the above general formula (II) in the usual way. Forexample, the compounds represented by the above general formula (I) ofthe present invention can be prepared using aglucopyranosyloxybenzylbenzene derivative represented by the abovegeneral formula (II) according to the following procedure:

wherein X represents a leaving group such as a bromine atom and achlorine atom; and R and P have the same meanings as defined above.

A prodrug represented by the above general formula (I) can be preparedby protecting the hydroxy group of a glucopyranosyloxybenzylbenzenederivative represented by the above general formula (II) with a reagentfor protecting represented by the above general formula (III) in thepresence of a base such as pyridine, triethylamine,N,N-diisopropylethylamine, picoline, lutidine, collidine, quinuclidine,1,2,2,6,6-pentamethylpiperidine or 1,4-diazabicyclo[2.2.2]octane in aninert solvent or without any solvent. As the solvent used,dichloromethane, acetonitrile, ethyl acetate, diisopropyl ether,chloroform, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, acetone,tert-butanol, a mixed solvent thereof and the like can be illustrated.The reaction temperature is usually from −40° C. to reflux temperature,and the reaction time is usually from 30 minutes to 2 days, varyingbased on a used starting material, solvent and reaction temperature.

For example, the compounds represented by the above general formula (II)of the present invention which are used as starting materials in theaforementioned production process can be prepared according to thefollowing procedure:

wherein M represents a hydroxy-protective group; X¹ represents a leavinggroup such as a trichloroacetoimidoyloxy group, an acetoxy group, abromine atom or a fluorine atom; one of Y and Z represents MgBr, MgCl,MgI or a lithium atom, while the other represents a formyl group; and Rhas the same meaning as defined above.Process 1

A compound represented by the above general formula (VI) can be preparedby condensing a benzaldehyde derivative represented by the above generalformula (IV) with a Grignard reagent or a lithium reagent represented bythe above general formula (V), or by condensing a Grignard reagent or alithium reagent represented by the above general formula (IV) with abenzaldehyde derivative represented by the above general formula (V) inan inert solvent. As the solvent used, tetrahydrofuran, diethyl ether, amixed solvent thereof and the like can be illustrated. The reactiontemperature is usually from −78° C. to reflux temperature, and thereaction time is usually from 10 minutes to 1 day, varying based on aused starting material, solvent and reaction temperature.

Process 2

A compound represented by the above general formula (VII) can beprepared by subjecting a compound represented by the above generalformula (VI) to oxidation using a Dess-Martin reagent in an inertsolvent. As the solvent used, dichloromethane, chloroform, acetonitrile,a mixed solvent thereof and the like can be illustrated. The reactiontemperature is usually from 0° C. to reflux temperature, and thereaction time is usually from 1 hour to 1 day, varying based on a usedstarting material, solvent and reaction temperature.

Process 3

A compound represented by the above general formula (VIII) can beprepared by subjecting a compound represented by the above generalformula (VI) to catalytic hydrogenation using a palladium catalyst suchas palladium-carbon powder in the presence or absence of an acid such ashydrochloric acid in an inert solvent, and removing a protective groupin the usual way as occasion demands. As the solvent used in thecatalytic hydrogenation, methanol, ethanol, tetrahydrofuran,ethylacetate, acetic acid, isopropanol, a mixed solvent thereof and thelike can be illustrated. The reaction temperature is usually from roomtemperature to reflux temperature, and the reaction time is usually from30 minutes to 1 day, varying based on a used starting material, solventand reaction temperature. The compound of the above general formula(VIII) can be converted into a salt thereof such as a sodium salt or apotassium salt in the usual way.

Process 4

A compound represented by the above general formula (VIII) can beprepared by removing the protective group M of a compound represented bythe above general formula (VII) in the usual way, condensing theresulting compound with methyl chloroformate in the presence of a basesuch as triethylamine, diisopropylethylamine or4-(N,N-dimethylamino)pyridine in an inert solvent and subjecting theresulting carbonate compound to reduction using a reducing agent such assodium borohydride. As the solvent used in the condensing reaction,tetrahydrofuran, dichloromethane, acetonitrile, ethyl acetate, diethylether, a mixed solvent thereof and the like can be illustrated. Thereaction temperature is usually from 0° C. to reflux temperature, andthe reaction time is usually from 30 minutes to 1 day, varying based ona used starting material, solvent and reaction temperature. As thesolvent used in the reducing reaction, a mixed solvent withtetrahydrofuran and water, and the like can be illustrated. The reactiontemperature is usually from 0° C. to reflux temperature, and thereaction time is usually from 1 hour to 1 day, varying based on a usedstarting material, solvent and reaction temperature. The compound of theabove general formula (VIII) can be converted into a salt thereof suchas a sodium salt or a potassium salt in the usual way.

Process 5

A glucoside represented by the above general formula (X) can be preparedby subjecting a benzylphenol derivative represented by the above generalformula (VIII) or a salt thereof to glycosidation using a glycosyl-donorrepresented by the above general formula (IX) such as2,3,4,6-tetra-O-acetyl-1-O-trichloroacetoimidoyl-α-D-glucopyranose,1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose,2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide and2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl fluoride in the presence of anactivating reagent such as boron trifluoride diethyl ether complex,silver trifluoromethanesulfonate, tin(IV) chloride or trimethylsilyltrifluoromethanesulfonate in an inert solvent. As the solvent used,dichloromethane, toluene, acetonitrile, nitromethane, ethyl acetate,diethyl ether, chloroform, a mixed solvent thereof and the like can beillustrated. The reaction temperature is usually from −30° C. to refluxtemperature, and the reaction time is usually from 10 minutes to 1 day,varying based on a used starting meterial, solvent and reactiontemperature.

Process 6

A glucopyranosyloxybenzylbenzene derivative represented by the abovegeneral formula (II) can be prepared by subjecting a glucosiderepresented by the above general formula (X) to alkaline hydrolysis toremove the hydroxy-protective groups. As the solvent used, water,methanol, ethanol, tetrahydrofuran, a mixed solvent thereof and the likecan be illustrated, and as alkaline materials, sodium hydroxide, sodiummethoxide, sodium ethoxide or the like can be used. The treatmenttemperature is usually from 0° C. to reflux temperature, and thetreatment time is usually from 30 minutes to 6 hours, varying based on aused starting material, solvent and treatment temperature.

The compounds of the present invention obtained by the above productionprocess can be isolated and purified by conventional separation meanssuch as fractional recrystallization, purification using chromatography,solvent extraction and solid phase extraction.

The prodrugs represented by the above general formula (I) of the presentinvention include their hydrates and their solvates withpharmaceutically acceptable solvents such as ethanol.

The prodrug represented by the above general formula (I) of the presentinvention is converted into a glucopyranosyloxybenzylbenzene derivativerepresented by the above general formula (II) as an active form thereofin vivo and can exert an excellent inhibitory activity in human SGLT2.In addition, the prodrugs represented by the above general formula (I)of the present invention have an improved oral absorption, andpharmaceutical compositions comprising as the active ingredient theprodrug have a highly usefulness as oral formulations. Therefore, theprodrugs of the present invention are extremely useful as agents for theprevention or treatment of a disease associated with hyperglycemia suchas diabetes, diabetic complications, obesity or the like.

When the pharmaceutical compositions of the present invention areemployed in the practical treatment, various dosage forms are useddepending on their uses. As examples of the dosage forms, powders,granules, fine granules, dry sirups, tablets, capsules, injections,solutions, ointments, suppositories, poultices and the like areillustrated, which are orally or parenterally administered.

These pharmaceutical compositions can be prepared by admixing with or bydiluting and dissolving with an appropriate pharmaceutical additive suchas excipients, disintegrators, binders, lubricants, diluents, buffers,isotonicities, antiseptics, moistening agents, emulsifiers, dispersingagents, stabilizing agents, dissolving aids and the like, andformulating the mixture in accordance with pharmaceutically conventionalmethods depending on their dosage forms.

When the pharmaceutical compositions of the present invention areemployed in the practical treatment, the dosage of a compound of thepresent invention as the active ingredient is appropriately decideddepending on the age, sex, body weight and degrees of symptoms andtreatment of each patient, which is approximately within the range offrom 0.1 to 1,000 mg per day per adult human in case of oraladministration and approximately within the range of from 0.01 to 300 mgper day per adult human in case of parenteral administration, and thedaily dose can be divided into one to several doses per day andadministered suitably.

EXAMPLES

The present invention is further illustrated in more detail by way ofthe following Reference Examples, Examples and Test Examples. However,the present invention is not limited thereto.

Reference Example 1

2-(4-Isobutylbenzyl)phenol

A Grignard reagent was prepared from 2-benzyloxy-1-bromobenzene (0.20g), magnesium (0.026 g), a catalytic amount of iodine andtetrahydrofuran (1 mL) in the usual manner. The obtained Grignardreagent was added to a solution of 4-isobutylbenzaldehyde (0.16 g) intetrahydrofuran (2 mL), and the mixture was stirred at room temperaturefor 30 minutes. The reaction mixture was purified by columnchromatography on aminopropyl silica gel (eluent: tetrahydrofuran) togive a diphenylmethanol compound (0.23 g). The obtained diphenylmethanolcompound was dissolved in ethanol (3 mL) and concentrated hydrochloricacid (0.1 mL). To the solution was added a catalytic amount of 10%palladium-carbon powder, and the mixture was stirred under a hydrogenatmosphere at room temperature overnight. The catalyst was removed byfiltration, and the filtrate was concentrated under reduced pressure.The residue was purified by column chromatography on silica gel (eluent:dichloromethane/hexane=1/1) to give 2-(4-isobutylbenzyl)phenol (0.10 g).

¹H-NMR (CDCl₃) δ ppm:

0.89 (6H, d, J=6.6 Hz), 1.75-1.90 (1H, m), 2.43 (2H, d, J=7.2 Hz), 3.97(2H, s), 4.66 (1H, s), 6.75-6.85 (1H, m), 6.85-6.95 (1H, m), 7.00-7.20(6H, m)

Reference Example 2

2-(4-Isopropoxybenzyl)phenol

The title compound was prepared in a similar manner to that described inReference Example 1 using 4-isopropoxybenzaldehyde instead of4-isobutylbenzaldehyde.

¹H-NMR (CDCl₃) δ ppm:

1.31 (6H, d, J=6.1 Hz), 3.93 (2H, s), 4.50 (1H, heptet, J=6.1 Hz), 4.72(1H, s), 6.75-6.85 (3H, m), 6.85-6.95 (1H, m), 7.05-7.20 (4H, m)

Reference Example 3

2-(4-Ethoxybenzyl)phenol

A Grignard reagent was prepared from 1-bromo-4-ethoxybenzene (1.5 g),magnesium (0.19 g), a catalytic amount of iodine and tetrahydrofuran (2mL) in the usual manner. To the obtained Grignard reagent solution wasadded dropwise a solution of 2-benzyloxybenzaldehyde (1.1 g) intetrahydrofuran (15 mL), and the mixture was stirred at room temperaturefor 30 minutes. To the reaction mixture were added a saturated aqueousammonium chloride solution (10 mL) and water (20 mL), and the mixturewas extracted with ethyl acetate (100 mL). The extract was washed withwater (20 mL) and brine (20 mL), and dried over anhydrous sodiumsulfate. Thereafter, the solvent was removed under reduced pressure. Theresidue was purified by column chromatography on silica gel (eluent:hexane/ethyl acetate=5/1) to give a diphenylmethanol compound (1.7 g).The obtained diphenylmethanol compound (1.7 g) was dissolved in ethanol(25 mL). To the solution were added concentrated hydrochloric acid (0.42mL) and a catalytic amount of 10% palladium-carbon powder, and themixture was stirred under a hydrogen atmosphere at room temperature for18 hours. The catalyst was removed by filtration, and the filtrate wasconcentrated under reduced pressure. To the residue was added ethylacetate (100 mL), and the mixture was washed with a saturated aqueoussodium hydrogen carbonate solution (30 mL) and brine (30 mL). Theorganic layer was dried over anhydrous sodium sulfate, and the solventwas removed under reduced pressure. The residue was purified by columnchromatography on silica gel (eluent: hexane/ethyl acetate=8/1) to give2-(4-ethoxybenzyl)phenol (0.85 g).

¹H-NMR (CDCl₃) δ ppm:

1.39 (3H, t, J=7.1 Hz), 3.93 (2H, s), 4.00 (2H, q, J=7.1 Hz), 4.72 (1H,s), 6.75-6.85 (3H, m), 6.85-6.95 (1H, m), 7.05-7.20 (4H, m)

Reference Example 4

2-(4-Ethylthiobenzyl)phenol

A Grignard reagent was prepared from 1-bromo-4-ethylthiobenzene (1.1 g),magnesium (0.12 g), a catalytic amount of iodine and tetrahydrofuran (5mL) in the usual manner. To the obtained Grignard reagent solution wasadded a solution of 2-(methoxymethoxy)benzaldehyde (0.56 g) intetrahydrofuran (12 mL), and the mixture was stirred at 65° C. for 10minutes. After cooling to ambient temperature, a saturated aqueousammonium chloride solution (5 mL) and water (20 mL) were added to thereaction mixture, and the mixture was extracted with ethyl acetate (80mL). The extract was washed with water (20 mL) and brine (20 mL), driedover anhydrous sodium sulfate, then the solvent was removed underreduced pressure. The residue was purified by column chromatography onsilica gel (eluent:hexane/ethyl acetate=4/1) to give a diphenylmethanolcompound (0.91 g). The obtained diphenylmethanol compound (0.90 g) wasdissolved in dichloromethane (15 mL). To the solution was added aDess-Martin reagent(1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one) (1.5 g),and the mixture was stirred at 25° C. for 26 hours. To the reactionmixture were added diethyl ether (75 mL) and 1 mol/L aqueous sodiumhydroxide solution (30 mL), the mixture was stirred vigorously, and theorganic layer was separated. The organic layer was washed with 1 mol/Laqueous sodium hydroxide solution (30 mL), water (30 mL, 3 times) andbrine (30 mL), dried over anhydrous sodium sulfate, and the solvent wasremoved under reduced pressure. The residue was purified by columnchromatography on silica gel (eluent: hexane/ethyl acetate=15/1-9/1) toafford a ketone compound (0.82 g). A mixture of the obtained ketonecompound (0.81 g), p-toluenesulfonic acid monohydrate (0.10 g) andmethanol (14 mL) was stirred at 60° C. for 4 hours. After cooling toambient temperature, the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (eluent: hexane/ethyl acetate=15/1) to give a deprotected compound(0.69 g). The obtained deprotected compound (0.68 g) was dissolved intetrahydrofuran (11 mL), triethylamine (0.41 mL) and methylchloroformate (0.22 mL) were added to the solution, and the mixture wasstirred at 25° C. for 1 hour. Furthermore, triethylamine (0.1 mL) andmethyl chloroformate (0.061 mL) were added to the reaction mixture, andthe mixture was stirred for 30 minutes. The reaction mixture wasfiltered, and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in tetrahydrofuran (14 mL) and water (7 mL),sodium borohydride (0.40 g) was added to the solution, and the mixturewas stirred at 25° C. for 7 hours. To the reaction mixture was addeddropwise 1 mol/L hydrochloric acid (15 mL), and the mixture wasextracted with ethyl acetate (75 mL). The extract was washed with water(20 mL), a saturated aqueous sodium hydrogen carbonate solution (20 mL)and brine (20 mL), dried over anhydrous sodium sulfate, and the solventwas removed under reduced pressure. The residue was purified by columnchromatography on silica gel (eluent: hexane/ethyl acetate=8/1) to give2-(4-ethylthiobenzyl)phenol (0.62 g).

¹H-NMR (CDCl₃) δ ppm:

1.29 (3H, t, J=7.3 Hz), 2.90 (2H, q, J=7.3 Hz), 3.96 (2H, s), 4.62 (1H,s), 6.75-6.80 (1H, m), 6.85-6.95 (1H, m), 7.05-7.20 (4H, m), 7.20-7.30(2H, m)

Reference Example 5

2-(4-Methoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

To a solution of 2-(4-methoxybenzyl)phenol (46 mg) and2,3,4,6-tetra-O-acetyl-1-O-trichloroacetoimdoyl-α-D-glucopy ranose (0.13g) in dichloromethane (2 mL) was added boron trifluoride diethyl ethercomplex (0.033 mL), and the mixture was stirred at room temperature for1 hour. The reaction mixture was purified by column chromatography onaminopropyl silica gel (eluent: dichloromethane) to give2-(4-methoxybenzyl)phenyl 2,3,4,6-terta-O-acetyl-β-D-glucopyranoside(0.11 g).

¹H-NMR (CDCl₃) δ ppm:

1.91 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.08 (3H, s), 3.77 (3H, s),3.80-3.95 (3H, m), 4.17 (1H, dd, J=2.5, 12.2 Hz), 4.29 (1H, dd, J=5.5,12.2 Hz), 5.11 (1H, d, J=7.5 Hz), 5.10-5.25 (1H, m), 5.25-5.40 (2H, m),6.75-6.85 (2H, m), 6.95-7.10 (5H, m), 7.10-7.25 (1H, m)

Reference Example 6

2-(4-Methylbenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 5 using 2-(4-methylbenzyl)-phenol instead of2-(4-methoxybenzyl)phenol.

¹H-NMR (CDCl₃) δ ppm:

1.89 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.07 (3H, s), 2.30 (3H, s),3.80-3.95 (3H, m), 4.17 (1H, dd, J=2.5, 12.3 Hz), 4.28 (1H, dd, J=5.5,12.3 Hz), 5.11 (1H, d, J=7.5 Hz), 5.10-5.25 (1H, m), 5.25-5.40 (2H, m),6.90-7.20 (8H, m)

Reference Example 7

2-(4-Ethylbenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 5 using 2-(4-ethylbenzyl)-phenol instead of2-(4-methoxybenzyl)phenol.

¹H-NMR (CDCl₃) δ ppm:

1.20 (3H, t, J=7.6 Hz), 1.87 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.08(3H, s), 2.60 (2H, q, J=7.6 Hz), 3.80-4.00 (3H, m), 4.18 (1H, dd, J=2.3,12.2 Hz), 4.28 (1H, dd, J=5.4, 12.2 Hz), 5.11 (1H, d, J=7.5 Hz),5.10-5.25 (1H, m), 5.25-5.40 (2H, m), 6.90-7.25 (8H, m)

Reference Example 8

2-(4-Isobutylbenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 5 using 2-(4-isobutylbenzyl)phenol instead of2-(4-methoxybenzyl)phenol.

¹H-NMR (CDCl₃) δ ppm:

0.88 (6H, d, J=6.6 Hz), 1.75-1.90 (1H, m), 1.87 (3H, s), 2.03 (3H, s),2.05 (3H, s), 2.08 (3H, s), 2.42 (2H, d, J=7.2 Hz), 3.80-3.95 (3H, m),4.18 (1H, dd, J=2.4, 12.3 Hz), 4.29 (1H, dd, J=5.5, 12.3 Hz), 5.11 (1H,d, J=7.6 Hz), 5.10-5.25 (1H, m), 5.25-5.40 (2H, m), 6.90-7.25 (8H, m)

Reference Example 9

2-(4-Ethoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 5 using 2-(4-ethoxybenzyl)phenol instead of2-(4-methoxybenzyl)phenol.

¹H-NMR (CDCl₃) δ ppm:

1.39 (3H, t, J=7.0 Hz), 1.91 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.07(3H, s), 3.80-3.95 (3H, m), 3.99 (2H, q, J=7.0 Hz), 4.18 (1H, dd, J=2.5,12.3 Hz), 4.28 (1H, dd, J=5.6, 12.3 Hz), 5.10 (1H, d, J=7.7 Hz),5.15-5.25 (1H, m), 5.25-5.40 (2H, m), 6.75-6.85 (2H, m), 6.95-7.10 (5H,m), 7.10-7.20 (1H, m)

Reference Example 10

2-(4-Isopropoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 5 using 2-(4-isopropoxybenzyl)phenol instead of2-(4-methoxybenzyl)phenol.

¹H-NMR (CDCl₃) δ ppm:

1.30 (6H, d, J=6.0 Hz), 1.90 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.08(3H, s), 3.80-3.90 (3H, m), 4.18 (1H, dd, J=2.3, 12.3 Hz), 4.28 (1H, dd,J=5.5, 12.3 Hz), 4.48 (1H, heptet, J=6.0 Hz), 5.10 (1H, d, J=7.7 Hz),5.10-5.25 (1H, m), 5.25-5.40 (2H, m), 6.70-6.85 (2H, m), 6.90-7.10 (5H,m), 7.10-7.20 (1H, m)

Reference Example 11

2-(4-Methoxybenzyl)phenyl β-D-glucopyranoside

Sodium methoxide (28% methanol solution; 0.12 mL) was added to asolution of 2-(4-methoxybenzyl)phenyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (0.11 g) in methanol (4 mL),and the mixture was stirred at room temperature for 30 minutes. Thesolvent was removed under reduced pressure. The residue was purified bycolumn chromatography on silica gel (eluent:dichloromethane/methanol=10/1) to give 2-(4-methoxybenzyl)-phenylβ-D-glucopyranoside (65 mg).

¹H-NMR (CD₃OD) δ ppm:

3.35-3.55 (4H, m), 3.69 (1H, dd, J=5.1, 12.1 Hz), 3.73 (3H, s),3.80-4.00 (2H, m), 4.03 (1H, d, J=15.1 Hz), 4.91 (1H, d, J=7.4 Hz),6.75-6.85 (2H,m), 6.85-6.95 (1H, m), 6.95-7.10 (1H, m), 7.10-7.20 (4H,m)

Reference Example 12

2-(4-Methylbenzyl)phenyl β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 11 using 2-(4-methylbenzyl)-phenyl2,3,4,6-tetra-0-acetyl-β-D-glucopyranoside instead of2-(4-methoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

¹H-NMR (CD₃OD) δ ppm:

2.27 (3H, s), 3.35-3.55 (4H, m), 3.69 (1H, dd, J=5.2, 12.0 Hz),3.80-3.90 (1H, m), 3.94 (1H, d, J=15.0 Hz), 4.05 (1H, d, J=15.0 Hz),4.85-4.95 (1H, m), 6.85-6.95 (1H, m), 6.95-7.20 (7H, m)

Reference Example 13

2-(4-Ethylbenzyl)phenyl β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 11 using 2-(4-ethylbenzyl)-phenyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside instead of2-(4-methoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

¹H-NMR (CD₃OD) δ ppm:

1.15-1.25 (3H, m), 2.50-2.65 (2H, m), 3.35-3.55 (4H, m), 3.65-3.75(1H,m), 3.80-4.00 (2H, m), 4.06 (1H, d, J=14.9 Hz), 4.85-5.00 (1H, m),6.85-7.00 (1H, m), 7.00-7.20 (7H, m)

Reference Example 14

2-(4-Isobutylbenzyl)phenyl β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 11 using 2-(4-isobutylbenzyl)phenyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside instead of2-(4-methoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

¹H-NMR (CD₃OD) δ ppm:

0.80-0.95 (6H, m), 1.70-1.90 (1H, m), 2.41 (2H, d, J=7.1 Hz), 3.30-3.55(4H, m), 3.60-3.75 (1H, m), 3.80-3.95 (1H, m), 3.95 (1H, d, J=15.0 Hz),4.06 (1H, d, J=15.0 Hz), 4.85-4.95 (1H, m), 6.80-7.20 (8H, m)

Reference Example 15

2-(4-Ethoxybenzyl)phenyl β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 11 using 2-(4-ethoxylbenzyl)phenyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside instead of2-(4-methoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

¹H-NMR (CD₃OD) δ ppm:

1.35 (3H, t, J=6.8 Hz), 3.35-3.55 (4H, m), 3.60-3.75 (1H, m), 3.80-4.10(5H, m), 4.90 (1H, d, J=7.1 Hz), 6.70-6.85 (2H, m), 6.85-6.95 (1H, m),7.00-7.20 (5H, m)

Reference Example 16

2-(4-Isopropoxybenzy)phenyl β-D-Glucopyranoside

The title compound was prepared in a similar manner to that described inReference Example 11 using 2-(4-isopropoxylbenzyl)phenyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside instead of2-(4-methoxybenzyl)phenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

¹H-NMR (CD₃OD) δ ppm:

1.27 (6H, d, J=6.0 Hz), 3.35-3.55 (4H, m), 3.69 (1H, dd, J=5.4, 12.1Hz), 3.88 (1H, dd, J=2.0, 12.1 Hz), 3.91 (1H, d, J=15.0 Hz), 4.02 (1H,d, J=15.0 Hz), 4.51 (1H, heptet, J=6.0 Hz), 4.91 (1H, d, J=7.7 Hz),6.70-6.85 (2H,m), 6.85-6.95 (1H, m), 7.00-7.10 (1H, m), 7.10-7.20 (4H,m)

Reference Example 17

2-(4-Ethylthiobenzyl)phenyl β-D-glucopyranoside

To a solution of 2-(4-ethylthiobenzyl)phenol (0. 51 g) and1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose (2.4 g) in toluene (6.3 mL)and dichloromethane (2.7 mL) was added boron trifluoride diethyl ethercomplex (0.78 mL), and the mixture was stirred at room temperature for 9hours. To the reaction mixture were added ethyl acetate (70 mL) and asaturated aqueous sodium hydrogen carbonate solution (25 mL), and theorganic layer was separated. The organic layer was washed with brine (25mL), dried over anhydrous sodium sulfate, and the solvent was removedunder reduced pressure. The residue was dissolved in methanol (10.5 mL),sodium methoxide (28% methanol solution; 0.08 mL) was added to thesolution, and the mixture was stirred at 25° C. for 18 hours. To thereaction mixture were added ethyl acetate (75 mL) and water (20 mL), andthe organic layer was separated. The organic layer was washed with brine(20 mL), dried over anhydrous sodium sulfate, and the solvent wasremoved under reduced pressure. The residue was purified by columnchromatography on silica gel (eluent: dichloromethane/methanol=10/1).The solvent was removed under reduced pressure, diethyl ether was addedto the residue, and the resulting precipitates were collected byfiltration. The obtained colorless solid was washed with diethyl etherand dried under reduced pressure to give 2-(4-ethylthiobenzyl)phenylβ-D-glucopyranoside (0.51 g).

¹H-NMR (CD₃OD) δ ppm:

1.24 (3H, t, J=7.3 Hz), 2.88 (2H, q, J=7.3 Hz), 3.35-3.55 (4H, m), 3.69(1H, dd, J=5.0, 12.2 Hz), 3.88 (1H, dd, J=2.0, 12.2 Hz), 3.95 (1H, d,J=15.1 Hz), 4.08 (1H, d, J=15.1 Hz), 4.91 (1H, d, J=7.3 Hz), 6.85-7.00(1H, m), 7.00-7.10 (1H, m), 7.10-7.30 (6H, m)

Example 1

2-(4-Methoxybenzyl)phenyl 6-O-ethoxycarbonyl-β-D-glucopyranoside

To a solution of 2-(4-methoxybenzyl)phenyl β-D-glucopyranoside (0.075 g)in 2,4,6-trimethylpyridine (2 mL) was added ethyl chloroformate (0.04mL) at room temperature. After the mixture was stirred at roomtemperature for 16 hours, a saturated aqueous citric acid solution wasadded to the reaction mixture, and the mixture was extracted with ethylacetate. The extract was washed with water and dried over anhydrousmagnesium sulfate, and the solvent was removed under reduced pressure.The residue was purified by preparative thin layer chromatography onsilica gel (eluent: dichloromethane/methanol=10/1) to give amorphous2-(4-methoxybenzyl)phenyl 6-O-ethoxycarbonyl-β-D-glucopyranoside (0.032g).

¹H-NMR (CD₃OD) δ ppm:

1.23 (3H, t, J=7.1 Hz), 3.30-3.65 (4H, m), 3.74 (3H, s), 3.93 (1H, d,J=15.1 Hz), 4.02 (1H, d, J=15.1 Hz), 4.05-4.20 (2H, m), 4.29 (1H, dd,J=6.4,11.7 Hz), 4.45 (1H, dd, J=2.2, 11.7 Hz), 4.89 (1H, d, J=7.4 Hz),6.75-6.85(2H, m), 6.85-7.05 (2H, m), 7.05-7.2 (4H, m)

Example 2

2-(4-Methoxybenzyl)phenyl 6-O-methoxycarbonyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 1 using methyl chloroformate instead of ethyl chloroformate.

¹H-NMR (CD₃OD) δ ppm:

3.30-3.65 (4H, m), 3.71 (3H, s), 3.74 (3H, s), 3.93 (1H, d, J=15.1 Hz),4.01 (1H, d, J=15.1 Hz), 4.30 (1H, dd, J=6.4, 11.7 Hz), 4.45 (1H, dd,J=2.1, 11.7 Hz), 4.89 (1H, d, J=7.4 Hz), 6.75-6.85 (2H, m), 6.85-7.05(2H, m), 7.05-7.20 (4H, m)

Example 3

2-(4-Methoxybenzyl)phenyl6-O-[2-(methoxy)ethyloxycarbonyl]-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 1 using 2-(methoxy)ethyl chloroformate instead of ethylchloroformate.

¹H-NMR (CD₃OD) δ ppm:

3.30-3.65 (9H, m), 3.74 (3H, s), 3.92 (1H, d, J=15.1 Hz), 4.02 (1H, d,J=15.1 Hz), 4.10-4.25 (2H, m), 4.30 (1H, dd, J=6.3, 11.7), 4.47 (1H, dd,J=2.1, 11.7 Hz), 4.89 (1H, d, J=7.4 Hz), 6.70-6.85 (2H, m), 6.85-7.05(2H, m), 7.05-7.20 (4H, m)

Example 4

2-(4-Methoxybenzyl)phenyl 6-O-hexanoyl-β-D-glucopyranoside

To a solution of 2-(4-methoxybenzyl)phenyl β-D-glucopyranoside (0.10 g)in 2,4,6-trimethylpyridine (2 mL) was added. hexanoyl chloride (0.072 g)at 0° C., and the mixture was stirred for 3 hours. To the reactionmixture was added 10% aqueous citric acid solution, and the mixture wasextracted with ethyl acetate. The organic layer was washed with 10%aqueous citric acid solution and brine. The organic layer was dried overanhydrous magnesium sulfate, and the solvent was removed under reducedpressure. The residue was purified by preparative thin layerchromatography on silica gel (eluent: dichloromethane/methanol=10/1) togive 2-(4-methoxybenzyl)phenyl 6-O-hexanoyl-β-D-glucopyranoside (0.030g).

¹H-NMR (CD₃OD) δ ppm:

0.80-0.95 (3H, m), 1.20-1.35 (4H, m), 1.50-1.65 (2H, m), 2.25-2.35(2H,m), 3.30-3.65 (4H, m), 3.74 (3H, s), 3.93 (1H, d, J=15.1 Hz), 4.01(1H, d, J=15.1 Hz), 4.22 (1H, dd, J=6.7, 11.8 Hz), 4.42 (1H, dd, J=2.2,11.8 Hz), 4.85-4.95 (1H, m), 6.75-6.85 (2H, m), 6.85-7.05 (2H, m),7.05-7.20 (4H, m)

Example 5

2-(4-Methoxybenzyl)phenyl 6-O-propionyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 4 using propionyl chloride instead of hexanoyl chloride.

¹H-NMR (CD₃OD) δ ppm:

1.08 (3H, t, J=7.6 Hz), 2.25-2.40 (2H, m), 3.30-3.55 (3H, m), 3.55-3.65(1H, m), 3.74 (3H, s), 3.93 (1H, d, J=15.1 Hz), 4.01 (1H, d, J=15.1 Hz),4.23 (1H, dd, J=6.7, 11.8 Hz), 4.40 (1H, dd, J=2.1, 11.8 Hz), 4.85-4.95(1H, m), 6.75-6.85 (2H, m), 6.85-7.05 (2H, m), 7.05-7.20 (4H, m)

Example 6

2-(4-Methoxybenzyl)phenyl 6-O-butyryl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 4 using butyryl chloride instead of hexanoyl chloride.

¹H-NMR (CD₃OD) δ ppm:

0.90 (3H, t, J=7.4 Hz), 1.50-1.70 (2H, m), 2.20-2.35 (2H, m), 3.30-3.65(4H, m), 3.74 (3H, s), 3.93 (1H, d, J=15.1 Hz), 4.01 (1H, d, J=15.1 Hz),4.22 (1H, dd, J=6.7, 11.8 Hz), 4.42 (1H, dd, J=2.2, 11.8 Hz), 4.85-4.95(1H, m), 6.75-6.85 (2H, m), 6.85-7.05 (2H, m), 7.05-7.20 (4H, m)

Example 7

2-(4-Methoxybenzyl)phenyl 6-O-acetyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 4 using acetyl chloride instead of hexanoyl chloride.

¹H-NMR (CD₃OD) δ ppm:

2.02 (3H, s), 3.30-3.65 (4H, m), 3.74 (3H, s), 3.93 (1H, d, J=15.1 Hz),4.01 (1H, d, J=15.1 Hz), 4.24 (1H, dd, J=6.5, 11.9 Hz), 4.38 (1H, dd,J=2.2, 11.9 Hz), 4.85-4.95 (1H, m), 6.75-6.85 (2H, m), 6.85-7.05 (2H,m), 7.05-7.20 (4H, m)

Example 8

2-(4-Methoxybenzyl)phenyl 6-O-isobutyryl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 4 using isobutyryl chloride instead of hexanoyl chloride.

¹H-NMR (CD₃OD) δ ppm:

1.11 (3H, d, J=7.0 Hz), 1.12 (3H, d, J=7.0 Hz), 2.45-2.60 (1H, m),3.30-3.65 (4H, m), 3.74 (3H, s), 3.93 (1H, d, J=15.1 Hz), 4.00 (1H, d,J=15.1 Hz), 4.19 (1H, dd, J=6.9, 11.8 Hz), 4.43 (1H, dd, J=2.1, 11.8Hz), 4.85-4.95(1H, m), 6.75-6.85 (2H, m), 6.85-7.05 (2H, m), 7.05-7.20(4H, m)

Example 9

2-(4-Methoxybenzyl)phenyl 6-O-ethylsuccinyl-β-D-glucopyranoside

The title compound was prepared in a similar manner to that described inExample 4 using ethylsuccinyl chloride instead of hexanoyl chloride.

¹H-NMR (CD₃OD) δ ppm:

1.19 (3H, t, J=7.1 Hz), 2.50-2.70 (4H, m), 3.30-3.65 (4H, m), 3.74 (3H,s), 3.93 (1H, d, J=15.1 Hz), 4.02 (1H, d, J=15.1 Hz), 4.08 (2H, q, J=7.1Hz), 4.22 (1H, dd, J=6.7, 11.8 Hz), 4.44 (1H, dd, J=2.1, 11.8 Hz),4.85-4.95(1H, m), 6.75-7.25 (8H, m)

Example 10

2-(4-Methoxybenzyl)phenyl 6-O-isopropyloxycarbonyl-β-D-glucopyranoside

To a solution of isopropanol (0.12 g) in 2,4,6-trimethylpyridine (2 mL)was added triphosgene (0.022 g) at 0° C., and the mixture was stirredfor 1 hour. Thereafter, 2-(4-methoxybenzyl)phenyl β-D-glucopyranoside(0.075 g) was added to the reaction mixture, and the mixture was stirredat room temperature overnight. To the reaction mixture was added 10%aqueous citric acid solution, and the mixture was extracted with ethylacetate. The organic layer was washed with 10% aqueous citric acidsolution and water, and dried over magnesium sulfate, and the solventwas removed under reduced pressure. The residue was purified bypreparative thin layer chromatography on silica gel (eluent:dichloromethane/methanol=10/1) to give 2-(4-methoxybenzyl)-phenyl6-O-isopropyloxycarbonyl-β-D-glucopyranoside (0.024 g).

¹H-NMR (CD₃OD) δ ppm:

1.21 (3H, d, J=6.3 Hz), 1.23 (3H, d, J=6.3 Hz), 3.30-3.65 (4H, m), 3.74(3H, s), 3.93 (1H, d, J=15.1 Hz), 4.02 (1H, d, J=15.1 Hz), 4.28 (1H, dd,J=6.4, 11.7 Hz), 4.43 (1H, dd, J=2.2, 11.7 Hz), 4.70-4.85 (1H, m),4.85-4.95(1H, m), 6.75-7.20 (8H, m)

Examples 1-22

The compounds in the following Table 1 were prepared in a similar mannerto that described in Example 1 or 2 using a compound obtained inReference Examples 12-17. TABLE 1

Example R P 11 Methyl Ethoxycarbonyl 12 Methyl Methoxycarbonyl 13 EthylEthoxycarbonyl 14 Ethyl Methoxycarbonyl 15 Isobutyl Ethoxycarbonyl 16Isobutyl Methoxycarbonyl 17 Ethoxy Ethoxycarbonyl 18 EthoxyMethoxycarbonyl 19 Isopropyl Ethoxycarbonyl 20 Isopropyl Methoxycarbonyl21 Ethylthio Ethoxycarbonyl 22 Ethylthio Methoxycarbonyl

Test Example 1

Assay for Inhibitory Effect on Human SGLT2 Activity

1) Construction of the Plasmid Vector Expressing Human SGLT2

Preparation of the cDNA library for PCR amplification was performed byreverse transcription of a total RNA deprived from human kidney (Origene) with oligo dT as the primer, using SUPERSCRIPT PreamplificationSystem (Gibco-BRL: LIFE TECHNOLOGIES). The DNA fragment coding for humanSGLT2 was amplified by the Pfu DNA Polymerase (Stratagene)-used PCRreaction, in which the human kidney cDNA library described above wasused as the template and the following oligo nucleotides 0702F and0712R, presented as Sequence Numbers 1 and 2 respectively, were used asthe primers. The amplified DNA fragment was ligated into pCR-Blunt(Invitrogen), a vector for cloning, according to standard method of thekit. The competent cell, Escherichia coli HB101 (Toyobo), wastransformed according to usual method and then selection of thetransformants was performed on the LB agar medium containing 50 μg/mL ofkanamycin. After the plasmid DNA was extracted and purified from the oneof the transformants, amplifying of the DNA fragment coding for humanSGLT2 was performed by the Pfu DNA Polymerase (Stratagene)-used PCRreaction, in which the following oligo nucleotides 0714F and 0715R,presented as Sequence Numbers 3 and 4 respectively, were used as theprimers. The amplified DNA fragment was digested with restrictionenzymes, Xho I and Hind III, and then purified with Wizard PurificationSystem (Promega). This purified DNA fragment was inserted at thecorresponding multi-cloning sites of pcDNA3.1 (−) Myc/His-B(Invitrogen), a vector for expressing of fusion protein. The competentcell, Escherichia coli HB101 (Toyobo), was transformed After the genetransfer, the cells were harvested by centrifugation and resuspendedwith OPTI-MEM I medium (1 mL/cuvette). To each well in 96-wells plate,125 μL of this cell suspension was added. After overnight culture at 37°C. under 5% CO₂, 125 μL of DMEM medium which is containing 10% of fetalbovine serum (Sanko Jyunyaku), 100 units/mL sodium penicillin G(Gibco-BRL: LIFE TECHNOLOGIES) and 100 μg/mL streptomycin sulfate(Gibco-BRL: LIFE TECHNOLOGIES) was added to each well. After a cultureuntil the following day, these cells were used for the measurement ofthe inhibitory activity against the uptake ofmethyl-α-D-glucopyranoside.

3) Measurement of the Inhibitory Activity Against the Uptake ofMethyl-α-D-glucopyranoside

After a test compound was dissolved in dimethyl sulfoxide and dilutedwith the uptake buffer (a pH 7.4 buffer containing 140 mM sodiumchloride, 2 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesiumchloride, 5 mM methyl-α-D-glucopyranoside, 10 mM2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid and 5 mM tris(hydroxymethyl) aminomethane), each diluent was used as test sample formeasurement of the inhibitory activity. After removal of the medium ofthe COS-7 cells expressing transiently human SGLT2, to each well 200 μLof the pretreatment buffer (a pH 7.4 buffer containing 140 mM cholinechloride, 2 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesiumchloride, 10 mM 2-[4-(2-hydroxyethyl)-1piperazinyl]-ethane sulfonic acidand 5 mM tris(hydroxymethyl)aminomethane) according to usual method andthen selection of the transformant was performed on the LB agar mediumcontaining 100 μg/mL of ampicillin. After the plasmid DNA was extractedand purified from this transformant, the base sequence of the DNAfragment inserted at the multi-cloning sites of pcDNA3.1 (−) Myc/His-Bwas analyzed. This clone had a single base substitution (ATC which codesfor the isoleucine-433 was substituted by GTC) compared with the humanSGLT2 reported by Wells et al (Am. J. Physiol., Vol. 263, pp. 459-465(1992)). Sequentially, a clone in which valine is substituted for theisoleucine-433 was obtained. This plasmid vector expressing human SGLT2in which the peptide presented as Sequence Number 5 is fused to thecarboxyl terminal alanine residue was designated KL29. Sequence Number 1ATGGAGGAGCACACAGAGGC Sequence Number 2 GGCATAGAAGCCCCAGAGGA SequenceNumber 3 AACCTCGAGATGGAGGAGCACACAGAGGC Sequence Number 4AACAAGCTTGGCATAGAAGCCCCAGAGGA Sequence Number 5KLGPEQKLISEEDLNSAVDHHHHHH2) Preparation of the Cells Expressing Transiently Human SGLT2

KL29, the plasmid coding human SGLT2, was trnasfected into COS-7 cells(RIKEN CELL BANK RCB0539) by electroporation. Electroporation wasperformed with GENE PULSER II (Bio-Rad Laboratories) under thecondition: 0.290 kV, 975 μF, 2×10⁶ cells of COS-7 cell and 20 μg of KL29in 500 μL of OPTI-MEM I medium (Gibco-BRL: LIFE TECHNOLOGIES) in the 0.4cm type cuvette. was added, and the cells were incubated at 37° C. for10 minutes. After the pretreatment buffer was removed, 200 μL of thesame buffer was added again, and the cells were incubated at 37° C. for10 minutes. The buffer for measurement was prepared by adding of 7 μL ofmethyl-α-D-(U-14C)glucopyranoside (Amersham Pharmacia Biotech) to 525 μLof the prepared test sample. For the control, the buffer for measurementwithout test compound was prepared. For estimate of the basal uptake inthe absence of test compound and sodium, the buffer for measurement ofthe basal uptake, which contains 140 mM choline chloride in place ofsodium chloride, was prepared similarly. After the pretreatment bufferwas removed, 75 μL of the buffer for measurement was added to each well,and the cells were incubated at 37° C. for 2 hours. After the buffer formeasurement was removed, 200 μL of the washing buffer (a pH 7.4 buffercontaining 140 mM choline chloride, 2 mM potassium chloride, 1 mMcalcium chloride, 1 mM magnesium chloride, 10 mMmethyl-α-D-glucopyranoside, 1 mM2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid and 5 mMtris(hydroxymethyl)aminomethane) was added to each well and immediatelyremoved. After two additional washing, the cells were solubilized byaddition of 75 μL of 0.2 N sodium hydroxide to each well. After the celllysates were transferred to the PicoPlate (Packard) and 150 μL ofMicroScint-40 (Packard) was added, the radioactivity was measured withmicroplate scintillation counter TopCount (Packard). The difference inuptake was obtained as 100% value by subtracting the radioactivity inthe basal uptake from that in control and then the concentrations atwhich 50% of uptake was inhibited (IC₅₀ Value) were calculated from theconcentration-inhibition curve by least square method. The results areshown in the following Table 2. TABLE 2 Test compound IC₅₀ value (nM)Reference Example 11 350 Reference Example 12 450 Reference Example 13140 Reference Example 14 500 Reference Example 15 330 Reference Example16 370 Reference Example 17 110

Test Example 2

Assay for Oral Absorbability

1) Preparation of the Samples for Measurement of the Drug ConcentrationAfter Intravenous Injection to the Tail Vein

As experimental animal, overnight fasted SD rats (CLEA JAPAN, INC.,male, 5 weeks of age, 140-170 g) were used. Sixty mg of a test compoundwas suspended or dissolved in 1.8 mL of ethanol and then dissolved byadding 7.2 mL of polyethylene glycol 400 and 9 mL of saline to prepare a3.3 mg/mL solution. The body weights of rats were measured and then thesolution of the test compound was intravenously injected to the tailvein of unanesthetized rats at the dose of 3 mL/kg (10 mg/kg). Theintravenous injection to the tail was performed with 26 G injectionneedle and 1 mL syringe. The sampling times for collection of blood were2, 5, 10, 20, 30, 60 and 120 minutes after the intravenous injection tothe tail vein. The blood was centrifuged and the plasma was used as thesample for measurement of the drug concentration in blood.

2) Preparation of the Samples for Measurement of the Drug ConcentrationAfter Oral Administration

As experimental animal, overnight fasted SD rats (CLEA JAPAN, INC.,male, 5 weeks of age, 140-170 g) were used. A test compound wassuspended or dissolved in 0.5% sodium carboxymethylcellulose solution atthe concentration of 1 mg/mL of active form. After the body weights ofrats were measured, the liquid containing the test compound describedabove was orally administered at the dose of 10 mL/kg (10 mg/kg asactive form). The oral administration was performed with gastric tubefor rat and 2.5 mL syringe. The sampling times for collection of bloodwere 15, 30, 60, 120 and 240 minutes after the oral administration. Theblood was centrifuged and the plasma was used as the sample formeasurement of the drug concentration in blood.

3) Measurement of Drug Concentration

To 0.1 mL of the plasma obtained in 1) and 2) described above, 1 μg of2-(4-ethoxybenzyl)phenyl β-D-glucopyranoside described in ReferenceExample 15 was added as internal standard and then deproteinization wasperformed by adding 1 mL of methanol. After centrifugation, the methanolphase was evaporated to dryness under a stream of nitrogen. The residuewas dissolved in 300 μL of the mobile phase and a 30 μL aliquot of thesolution was injected into HPLC. The drug concentration in plasma wasanalysed by HPLC method under the condition as follows.

Column : Inertsil ODS-2 (4.6×250 mm)

Mobile Phase: Acetonitrile/10 mM Phosphate buffer (pH 3.0)=25:75(v/v)

Column Temperature: 50° C.

Flow Rate: 1.0 mL/minute

Wavelength for Measurement: UV 232 nm

After addition of 1 μg of 2-(4-ethoxybenzyl)phenyl β-D-glucopyranosidedescribed in Reference Example 15 as internal standard and eachconcentration (1.0, 0.5, 0.2, 0.1, 0.05 and 0.02 μg) of2-(4-methoxybenzyl)phenyl β-D-glucopyranoside described in ReferenceExample 11 to 0.1 mL of the blank plasma, similar operating describedabove was performed and then the standard curve was prepared.

Each area under the plasma concentration-time curve by intravenousinjection to the tail vein and oral administration of test compound wasestimated with WinNonlin Standard made by Pharsight Corporation from theplasma concentrations at each time obtained from HPLC and then thebioavailability (%) was calculated by the formula as follows. Theresults are shown in the following Table 3.${{Bioavailability}\quad(\%)} = {\frac{\begin{matrix}{{Area}\quad{under}\quad{the}\quad{Plasma}\quad{Concentration}\text{-}} \\{{Time}\quad{Curve}\quad{by}\quad{Oral}\quad{Administration}}\end{matrix}}{\begin{matrix}{{Area}\quad{under}\quad{the}\quad{Plasma}\quad{Concentration}\text{-}} \\{{Time}\quad{Curve}\quad{by}\quad{Intravenous}\quad{Injection}} \\{{to}\quad{the}\quad{Tail}\quad{Vein}}\end{matrix}} \times 100}$ TABLE 3 Test compound Bioavailability (%)Example 1 46 Example 4 61 Reference Example 11 15

Test Example 3

Assay for the Facilitatory Effect on Urinary Glucose Excretion

As experimental animal, non-fasted SD rats (SLC. Inc., male, 8 weeks ofage, 270-320 g) were used. A test compound was suspended in 0.5%carboxymethyl solution and 0.3, 1 and 3 mg/mL suspension were prepared.After the body weights of rats were measured, the test suspension wasorally administered at the dose of 10 mL/kg (3, 10 and 30 mg/kg). Forthe control, just only 0.5% sodium carboxymethylcellulose solution wasorally administered at the dose of 10 mL/kg. The oral administration wasperformed with gastric tube for rat and 2.5 mL syringe. The head countin one group was 5 or 6. Collection of urine was performed in metaboliccage after the oral administration was finished. The sampling time forcollection of urine was 24 hours after the oral administration. Aftercollection of urine was finished, the urine volume was recorded and theurinary glucose concentration was measured. The glucose concentrationwas measured with a kit for laboratory test: Glucose B-Test WAKO (WakoPure Chemical Industries, Ltd.). The amount of urinary glucose excretionin 24 hours per 200 g of body weight was calculated from urine volume,urinary glucose concentration and body weight. The results are shown inthe following Table 4. TABLE 4 Amount of Urinary Glucose Dose ExcretionTest compound (mg/kg) (mg/24 hours · 200 g body weight) Example 1 3 5210 239 30 513

Test Example 4

Acute Toxicity Test

Four weeks old male ICR mice (CLEA JAPAN, INC. 22-28 g, 5 animals ineach group) were fasted for 4 hours, and 60 mg/mL of a suspension of atest compound in 0.5% carboxymethylcellulose solution was orallyadministered at the dose of 10 mL/kg(600 mg/kg). No death was observeduntil 24 hours after the administration as shown in the following Table5. TABLE 5 Test compound Death number Example 1 0/5Industrial Applicability

The glucopyranosyloxybenzylbenzene derivatives represented by the abovegeneral formula (I) of the present invention have an improved oralabsorption and can exert an excellent inhibitory activity in human SGLT2by converting into glucopyranosyloxybenzylbenzene derivativesrepresented by the above general formula (II) as active forms thereof invivo after oral administration. The present invention can provide agentsfor the prevention or treatment of a disease associated withhyperglycemia such as diabetes, diabetic complications, obesity or thelike, which are also suitable as oral formulations.

1-2. (canceled).
 3. A method as claimed in claim 14, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the generalformula:

wherein R¹ represents a lower alkyl group or a lower alkoxy group; andP¹ represents a lower acyl group, a lower alkoxy-substituted (loweracyl) group, a lower alkoxycarbonyl-substituted (lower acyl) group, alower alkoxycarbonyl group or a lower alkoxy-substituted (loweralkoxycarbonyl) group.
 4. A method as claimed in claim 14, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the generalformula:

wherein R represents a lower alkyl group, a lower alkoxy group, a loweralkylthio group, a lower alkoxy-substituted (lower alkyl) group, a loweralkoxy-substituted (lower alkoxy) group or a lower alkoxy-substituted(lower alkylthio) group; and P² represents a lower acyl group or a loweralkoxycarbonyl group.
 5. A method as claimed in claim 14, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the generalformula:

wherein R¹ represents a lower alkyl group or a lower alkoxy group; andP² represents a lower acyl group or a lower alkoxycarbonyl group.
 6. Amethod as claimed in claim 14, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the formula:


7. A method as claimed in claim 14, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the formula:

8-13. (canceled).
 14. A method for the prevention of a diseaseassociated with hyperglycemia, which comprises administering to apatient in need of the prevention of a disease associated withhyperglycemia a therapeutically effective amount of aglucopyranosyloxybenzylbenzene derivative represented by the generalformula:

wherein R represents a lower alkyl group, a lower alkoxy group, a loweralkylthio group, a lower alkoxy-substituted (lower alkyl) group, a loweralkoxy-substituted (lower alkoxy) group or a lower alkoxy-substituted(lower alkylthio) group; and P¹ represents a lower acyl group, a loweralkoxy-substituted (lower acyl) group, a loweralkoxycarbonyl-substituted (lower acyl) group, a lower alkoxycarbonylgroup or a lower alkoxy-substituted (lower alkoxycarbonyl) group. 15.(canceled).
 16. A method for providing a preventing effect on a diseaseassociated with hyperglycemia, which comprises administering to apatient in need of the preventing effect a therapeutically effectiveamount of a glucopyranosyloxybenzylbenzene derivative represented by thegeneral formula:

wherein R represents a lower alkyl group, a lower alkoxy group, a loweralkylthio group, a lower alkoxy-substituted (lower alkyl) group, a loweralkoxy-substituted (lower alkoxy) group or a lower alkoxy-substituted(lower alkylthio) group; and P¹ represents a lower acyl group, a loweralkoxy-substituted (lower acyl) group, a loweralkoxycarbonyl-substituted (lower acyl) group, a lower alkoxycarbonylgroup or a lower alkoxy-substituted (lower alkoxycarbonyl) group.
 17. Amethod as claimed in claim 16, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the generalformula:

wherein R¹ represents a lower alkyl group or a lower alkoxy group; andP¹ represents a lower acyl group, a lower alkoxy-substituted (loweracyl) group, a lower alkoxycarbonyl-substituted (lower acyl) group, alower alkoxycarbonyl group or a lower alkoxy-substituted (loweralkoxycarbonyl) group.
 18. A method as claimed in claim 16, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the generalformula:

wherein R represents a lower alkyl group, a lower alkoxy group, a loweralkylthio group, a lower alkoxy-substituted (lower alkyl) group, a loweralkoxy-substituted (lower alkoxy) group or a lower alkoxy-substituted(lower alkylthio) group; and P² represents a lower acyl group or a loweralkoxycarbonyl group.
 19. A method as claimed in claim 16, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the generalformula:

wherein R¹ represents a lower alkyl group or a lower alkoxy group; andP² represents a lower acyl group or a lower alkoxycarbonyl group.
 20. Amethod as claimed in claim 16, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the formula:


21. A method as claimed in claim 16, wherein theglucopyranosyloxybenzylbenzene derivative is represented by the formula: